This application is National Stage entry of International Application No. PCT/GB00/01261, filed Apr. 3, 2000, the entire specification claims and drawings of which are incorporated herewith by reference.
This invention relates to substituted azetidin-2-ones, to pharmaceutical compositions containing such compounds, and to their use in medicine as inhibitors of cysteine proteases, particularly the cathepsins.
The cathepsin family (C1) of lysosomal cysteine (or thiol) proteases is a subset of the papain superfamily (clan CA of cysteine proteases) and includes cathepsin B, H, K, S, L and the recently discovered cathepsins O, O2/K, V, X, Z and W (lymphopain). Related enzymes also regarded as cysteine proteases are the cytoplasmic Ca2+ dependent calpains (family C2). Cysteine proteases are classified both functionally and according to their active site, which has a thiol residue. They differ in substrate specificities and other enzymatic activities, these differences probably arising from evolutionary divergence.
The known cathepsins are synthesized on membrane bound ribosomes, transferred to the endoplasmic reticulum, then to the Golgi apparatus and finally to the lysosome and endosomes. They have an important function in regulation of intracellular protein metabolism, mobilisation of tissue proteins and conversion of proenzymes, prohormones and neuropeptides into biologically active molecules. The cathepsins are believed to be involved in a number of diseases.
Cathepsin K can be secreted into the extracellular space and is involved in bone and cartilage remodelling. Cathepsin K is implicated in the pathogenesis of osteoporosis. Cathepsin K inhibitors can prevent osteoporosis in animal models (PNAS. 1997. 94:14249-14254). Cathepsin L inhibitors have also been shown to inhibit osteoporosis (Bone, 1997. 20:465-471).
Cathepsin B and others have also been shown to be released extracellularly by various tumour cells and are thought to play a role in tumour invasion (Journal of cellular Physiology. 1992. 150:534-544).
The cathepsins have also been shown to play a role in rheumatoid arthotis (Arthritis and Rheumatism 1994. 37:236-247) and neuronal and cardiac ischaemia (European Journal of Neuroscience. 1998. 10.1723-1733).
Cathepsins S and L both play a role in the generation of free MHC class II molecules capable of binding antigenic peptides in the endosomes. These class II/peptide complexes move to the cell membrane and are involved in T lymphocyte activation. Inhibitors of Cathepsin S have been shown to inhibit allergic immune responses (Journal of Clinical Investigation. 1998. 101:2351-2363).
In addition to their role in the above diseases, cathepsins play a major role in the pathogenesis of infectious diseases. For example, cathepsins are used by the protozoal parasites Plasmodium (malaria) and Trypanosoma (Chagas Disease) to invade the human host and cathepsin inhibitors can inhibit experimental disease in both cases (Antimicrobial agents and chemotherapy. 1998. 42:2254-2258; Journal of Experimental Medicine. 1998. 188:725-734). Cathepsins are also virulence factors for several pathogenic bacteria.
A recent review (Annu. Rev. Physiol. 1997. 59:63-88) describes the state of the art of cysteine proteases, including the cathepsins, and their presumed biological functions. Another review (Exp. Opin. Ther. Patents, 1998, 8(6), pp645-672) deals with cathepsin B inhibitors as potential anti-metastatic agents.
International patent applications WO 96132408, WO 98/12176, WO 98/12210 and GB 9806287.0 describe, inter alia, classes of cysteine protease inhibitors which may be represented by formula (IA): 
wherein Y, R1, R2 and R3 represent the groups found in corresponding positions of the compounds disclosed in those publications. These known compounds are azetidin-2-ones which are monosubstituted at positions 3 and 4.
The present invention makes available a new class of cysteine protease inhibitors which differ in structure from those disclosed in WO 96/32408, WO 98/12176, WO 98/12210 and GB 9806287.0 principally in that they are disubstituted at the 3-position. These compounds are useful for the treatment of diseases mediated by cysteine protease activity, for example muscular dystrophy, osteoporosis, tumour metastasis, rheumatoid arthritis, neuronal or cardiac ischaemia, allergic immune response, and protozoal or bacterial disease.
According to the present invention, there is provided a compound of formula (I) 
Y represents xe2x80x94C(O)xe2x80x94 or xe2x80x94S(O2)xe2x80x94;
R represents an allyl (ie CH2xe2x95x90CHCH2xe2x80x94) group or a radical of formula R4-(ALK)p-(Z)n-(ALK)q- wherein Z represents xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94, ALK represents a divalent C1-C3alkyl or halogen-substituted C1-C3alkyl radical, R4 represents hydrogen or halogen, or an optionally substituted phenyl group, and n, p and q are independently 0 or 1, PROVIDED THAT (i) when R4 is hydrogen and both p and n are 0 then q is 1; and (ii) when R4 is halogen and n is 1 then p is 1; and (iii) when R4 is halogen then p, n and q are not all 0;
R1 represents xe2x80x94OCOR5, xe2x80x94OR5, xe2x80x94SR5, xe2x80x94S(O)R5, or xe2x80x94S(O)2R5;
R2 represents a radical of formula R6-(ALK)p-(Z)n-(ALK)q- wherein p, Z and ALK are as defined in relation to R, q is 0 or 1, n is 0 or 1 when q is 1 and n is 0 when q is 0, and R6 is hydrogen or an optionally substituted C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, cycloalkenyl, aryl or heterocyclic group; or R2 together with the carbon atom to which it is attached forms a cycloalkyl ring;
R3 represents xe2x80x94OR5 or xe2x80x94R5;
R5 represents a radical of formula R7xe2x80x94(A)txe2x80x94 wherein t is 0 or 1; A represents (i) an optionally substituted divalent C1-C6alkyl, radical which may be interrupted by one or more non-adjacent xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94 or xe2x80x94NHxe2x80x94 linkages, or (ii) a divalent C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, cycloalkenyl, aryl or heterocyclic radical, or (iii) a xe2x80x94NHxe2x80x94 link; and R7 represents hydrogen or an optionally substituted C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, cycloalkyl, cycloalkenyl, aryl or heterocyclic group;
or a pharmaceutically acceptable salt, hydrate or solvate thereof.
Pharmaceutically acceptable salts of the compounds of this invention include the sodium, potassium, magnesium, calcium, hydrogen chloride, tartaric acid, succinic acid, fumaric acid and p-toluenesulfonic acid salts.
Preferably, the R and R1 groups are cis to each other.
As used herein the term xe2x80x9c(C1-C6)alkylxe2x80x9d or xe2x80x9clower alkylxe2x80x9d means a straight or branched chain alkyl moiety having from 1 to 6 carbon atoms, including for example, methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylprop-1-yl, 2-methylprop-2-yl, pentyl, 3-methylbutyl, and hexyl. Similar terms such as xe2x80x9c(C1-C3)alkylxe2x80x9d are to be interpreted similarly.
As used herein the term xe2x80x9cC2-C6alkenylxe2x80x9d means a straight or branched chain alkenyl moiety having from 2 to 6 carbon atoms having at least one double bond of either E or Z stereochemistry where applicable. The term includes, for example, vinyl, allyl, 1- and 2-butenyl and 2-methyl-2-propenyl. Similar terms such as xe2x80x9c(C2-C3)alkenylxe2x80x9d are to be interpreted similarly.
As used herein the term xe2x80x9cC2-C6 alkynylxe2x80x9d means a straight chain or branched chain hydrocarbon groups having from two to six carbon atoms and having in addition one triple bond. This term would include for example, ethynyl, 1-propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl, Similar terms such as xe2x80x9c(C2-C3)alkynylxe2x80x9d are to be interpreted similarly.
As used herein the term xe2x80x9ccycloalkylxe2x80x9d means a saturated alicyclic moiety having from 3-7 carbon atoms and includes, for example, cyclohexyl, cycloheptyl, cyclopentyl, cyclobutyl and cyclopropyl.
As used herein the term xe2x80x9chalogenxe2x80x9d means fluoro, chloro, bromo or iodo.
As used herein the term xe2x80x9carylxe2x80x9d refers to a mono-, bi- or tri-cyclic, substituted or unsubstituted, carbocyclic aromatic group, and to groups consisting of two covalently linked substituted or unsubstituted monocyclic carbocyclic aromatic groups. Illustrative of such groups are phenyl, biphenyl and napthyl. Examples include C6-C12 aryl groups such as phenyl, biphenyl, naphthyl, tetrahydronaphthyl, dihydronaphthyl, and cyclohexyl phenyl.
As used herein the unqualified term xe2x80x9cheterocyclylxe2x80x9d or xe2x80x9cheterocyclicxe2x80x9d means a 5-7 membered heterocyclic ring, which may be aromatic or non-aromatic, containing one or more heteroatoms selected from S, N and O, and optionally fused to a benzene or hetero-atom containing ring. The term therefore includes C1-C11 heterocyclic groups containing 1-4 heteroatoms selected from nitrogen, sulfur or oxygen. Examples include thienyl, pyridyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3,4-tetrazolyl, imidazolyl, quinolinyl, isoquinolinyl, indolyl, pyrimidinyl, benzofuranyl, benzothienyl, morpholinyl, thiomorpholinyl, piperazinyl, piperidinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyridylphenyl, pyrimidylphenyl, pyrrolyl, furyl, thienyl, piperidinyl, imidazolyl, oxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, benzimrdazolyl, maleimido, succinimido, and phthalimido groups.
As used herein, the unqualified term xe2x80x9csubstitutedxe2x80x9d as applied to a group or radical means substituted with 1, 2, or 3 substituents selected from
(C1-C3)alkyl;
phenyl;
hydroxy or mercapto;
(C1-C3)alkoxy or (C1-C3)alkylthio;
phenoxy or phenylthio;
halogen;
trifluoromethyl;
nitro;
cyano (xe2x80x94CN);
carboxyl, and amidated, esterified or protected carboxyl;
amino, mono- or di-(C1-C3)alkylamino, or protected amino;
(C1-C3)alkylcarbonyl- or (C1-C3)alkylcarbonylamino-;
xe2x80x94CONHRA, xe2x80x94NHRA, xe2x80x94NRARB, or xe2x80x94CONRARB wherein RA and RB are independently (C1-C3)alkyl; and
xe2x80x94NHxe2x80x94C(xe2x95x90NR8)R9 wherein R9 is amino, mono- or di-(C1-C6)alkylamino, protected amino, or (C1-C3)alkyl, and R8 is hydrogen, (C1-C3)alkyl, or an N-protecting group.
As used herein the term xe2x80x9cprotecting groupxe2x80x9d when used in relation to an amino or carboxylic acid moeity in the compounds of this invention means a group which is used to render the amino or carboxylic acid moeity substantially non reactive, ie to neutralise its amino or carboxylic acid functionality. In this context, protected amino groups include amido and acylamino, protected hydroxy or mercapto groups include ethers and thioethers, protected carboxyl groups include esters, and imidazolyl, indolyl or guanidyl groups may be protected as t-butoxycarbonyl derivatives. These are only examples of the many protecting derivatives known in the art, and others will be known to the skilled man. Such protecting groups are of course well known, eg from the art of peptide synthesis, and are discussed in the widely used handbook by T. W. Greene and P. G. M. Wuts, Protective groups in Organic Synthesis, 2nd Edition, Wiley, New York 1991, and elsewhere in the chemical literature.
The azetidinone nucleus in the compounds of the invention has two asymmetric carbon atoms at position 3 (carrying the R group) and 4 (carrying the R1 group), and can exist as 4-diastereoisomers. While the invention includes all such diastereomers and mixtures thereof (including racemic mixtures), compounds in which the R and R1 groups are cis to each other are currently preferred, as are mixtures of diastereoisomers in which that configuration predominates.
As mentioned above, the compounds of the invention differ in structure from those of WO 96/32408, WO 98/12176, WO 98/12210 and GB 9806287.0 principally in that they carry a second substituent R at the 3-position of the azetidin-2-one ring. Thus the substituents R1, R2 and R3 in the compounds of the invention may be any of the groups falling within the above definitions of R1, R2 and R3 and which are present in corresponding positions of cysteine protease inhibitors disclosed in those patent applications. Without prejudice to the generality of the foregoing, in the compounds of the invention:
Y may be, for example, xe2x80x94C(O)xe2x80x94;
R may be, for example, allyl, methyl, ethyl, n-propyl, n-or iso-butyl, methyoxymethyl, ethoxymethyl, benzyl, or phenoxymethyl;
R1 may be, for example, acetoxy; butyloxy; 2-carboxyethyloxy; 2-aminoethyloxy; 2-fluoroethoxy; cyclopentyloxy; cyclohexyloxy; cyclohexylthio; phenoxy, phenoxy substituted by methyl, tert-butyl, trifluoromethyl, amino, hydroxy, acetamido, cyano, carboxy or fluoro; naphthyloxy; morpholino-phenyloxy; 2-hydroxyethylthio; phenylthio; phenylsulphonyl; 4-(2-carboxy-2-amino ethylyphenoxy; 2-pyridylthio; 4-pyridylthio; benzyloxy; 3-pyridyl-phenoxy; 3-tetrazolyl-phenoxy; 3,4-methylenedioxy-phenoxy; 3,4ethylenedioxy-phenoxy; tetrahydroquinolinoxy; quinolinoxy; or quinolinthio. Currently preferred are acetoxy and phenoxy.
R2 may be, for example, a phenyl group which may be substituted by one or more of hydroxy, halogen, methoxy, methyl, isopropyl, tert-butyl and trifluoromethyl; isopropyl, cyclohexyl; 3-pyridinyl; naphthyl; biphenyl; 2-thienyl; 3,4-methylenedioxyphenyl; 3,4-ethylenedioxy-phenyl; benzothienyl; thiazolyl; quinolinyl; isoquinolinyl; tetrahydroquinolinyl; tetrahydronaphthyl; aminonaphthyl; or acetamidonaphthyl. Presently preferred are phenyl, isopropyl, cyclohexyl and 3-pyridinyl.
R3 may be, for example, benzyloxy, 3-phenylpropyloxy, 3-phenylpropyl, 3-phenylprop-1-enyl, 6-N,N-dibenzyloxycarbonylguanidino-hexyl, 6-guanidino-hexyl, methoxy-methyleneoxy-methyl, 2-amino-ethoxy-methyl, 3-(pyridin-3- or 4-ylypropyl, or 3-(pyridin-3- or 4-ylprop-1-enyl.
Specific compounds of the invention include those of named and characterised in the Examples herein.
As stated, the compounds of the invention are inhibitors of cysteine proteases, for example cathepsins B, L, S and/or K. The invention therefore also provides a pharmaceutical composition containing a compound of formula (I) as defined above, and a pharmaceutically acceptable carrier. Also provided is the use of such a compound in the preparation of a composition for inhibiting cysteine protease activity in the body of a mammal suffering a disease mediated by such activity, and a method of treatment of an animal suffering from a disease mediated by cysteine protease activity, which method comprises administering to the mammal a sufficient amount of a compound of formula (I) as defined above to inhibit such activity.
Diseases mediated by cysteine protease activity include muscular dystrophy, osteoporosis, tumour metastasis, rheumatoid arthritis, neuronal or cardiac ischaemia, allergic immune response, and protozoal or bacterial disease.
Compositions with which the invention is concerned may be prepared for administration by any route consistent with the pharmacokinetic properties of the active ingredient(s).
Orally administrable compositions may be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions. Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants for example potato starch, or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia;
non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.
For topical application to the skin, the active ingredient(s) may be made up into a cream, lotion or ointment. Cream or ointment formulations, which may be used for the drug, are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.
The active ingredient(s) may also be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the drug can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anaesthetic, preservative and buffering agents can be dissolved in the vehicle. Intravenous infusion is another route of administration for the compounds.
Safe and effective dosages for different classes of patient and for different disease states will be determined by clinical trial as is required in the art. It will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
Compounds of the invention may be prepared by acylation of the 3amino group of a compound of formula (II) with an acylating derivative of a compound of formula (IlI) 
wherein Y, R, R1, R2 and R3 are as defined above except that any functional groups present in R, R1, R2 and R3 which might give rise to substantial amounts of unwanted by-products are protected, and thereafter removing any such protecting groups. In the acylation reaction, compound (III) may be activated as an active ester, for example the hydroxybenzotriazolyl ester, to facilitate the acylation reaction.
Compounds (II) are accessible from commercially available materials by widely known synthetic methods. Reaction Schemes 1 and 2 below illustrate synthetic routes to compounds (II) in which R is methyl, which may be modified as appropriate to produce other compounds of formula (II). Compounds (III) are in many cases commerially available, and otherwise are also accessible from commercially available materials by widely known synthetic methods. 
An example of an acylation reaction between a compound of formula (II) and a compound of formula (III) is shown in Scheme 3. In general, the amine (II) and the acid (III) are coupled either in presence of coupling reagent or by use of the chloride or anhydride of (III) in presence of base or activated ester.
In some cases, compounds of formula (I) may be prepared by coupling a compound of formula (IV) with a compound of formula (V): 
wherein Y1 represents xe2x80x94COxe2x80x94 or xe2x80x94S(O)2xe2x80x94, and R, R1, R2 and R3 are as defined above except that any functional groups present in R, R1, R2 and R3 which might give rise to substantial amounts of unwanted by-products are protected, and thereafter removing any such protecting groups. Here again the amine (IV) and the acid (V) (Y1xe2x95x90xe2x80x94COxe2x80x94) are coupled either in presence of coupling reagent or by use of the chloride or anhydride of (V) in presence of base, or by use of an activated ester.
Compounds (V) are in many cases commerially available, and otherwise are accessible from commercially available materials by widely known synthetic methods.
In some cases, one compound of formula (I) may be prepared from another of formula (I). For example, Scheme 3 shows a synthetic route in which the 4-acetoxy group in a compound of formula (I) wherein R1 is acetoxy is converted to a group xe2x80x94OR5 or xe2x80x94SR5. Conversion of the 4-acetoxy group is effected by reacting with R5XH in presence of lewis acids such as zinc acetate, zinc iodide, zinc chloride, titanium tetrachloride, palladium acetate, boron trifluoride, aluminium trichloride and the like or in presence of base such as sodium hydroxide. Reactive groups in R5 will of course be protected during such reactions, and subsequently deproteced. Thus, where a carboxy group is present in R5 it may be protected with diphenyl methyl or 1,1-dimethyl ethyl and an amino group in R5 may be protected with benzyloxycarbonyl or 1,1-dimethylethoxycarbonyl. Deprotection may be effected by hydrogenation or hydrolysis with acids. 
Monobactam derivatives of general formula I wherein R1 is xe2x80x94SR5 may be converted to, xe2x80x94SOR5, or xe2x80x94SO2R5 by oxidation with oxidizing agent such as m-chloroperbenzoic acid, hydrogen peroxide peracetic acid, potassium permanganate, or manganese dioxide.